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Evidence-BasedComplementaryandAlternativeMedicine
Volume2022,ArticleID7425538,10pages
/2022/7425538
ResearchArticle
The5-HTandPLCSignalingPathwaysRegulatetheSecretionof
IL-1β,TNF-αandBDNFfromNG2Cells
TingtingYang,1YueLi,1HuiWang,1PengShi,1LiuTeng,1HaiboGuo,1XiaohuaTu,1
andXinyuYao2
1SchoolofBasicMedicine,GuizhouUniversityofTraditionalChineseMedicine,Guiyang,China
2GraduateSchool,ChengdeMedicalCollege,Chengde,China
CorrespondenceshouldbeaddressedtoHuiWang;******@
Received4January2022;Revised24March2022;Accepted25March2022;Published13May2022
AcademicEditor:Lu´ısaMotadaSilva
Copyright©2022TingtingYangetal.isisanopenaccessarticledistributedundertheCreativeCommonsAttributionLicense,
whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.
epresentstudywasclariedtherelationshipbetweenNG2glialcellsand5-hydroxytryptamine(5-HT)tofurtherrevealedarole
intheregulationofcorticalexcitability.eco-localizationofNG2cellsand5-HTinratprefrontalcortexwasdeterminedusing
immunoerentconcentrationsof5--timePCRmeasuredtheex-
pressionofinterleukin-1β(IL-1β),tumornecrosisfactor-α(TNF-α)andbrain-derivedneurotrophicfactor(BDNF).Changesin
theexpressionofIL-1β,TNF-α,andBDNFinNG2cellsweredetectedaftertheadditionof5-HTreceptorspecicblockersand
phospholipaseC(PLC)specicactivatorsandinhibitors.eresultsconrmedthattheNG2proteinand5-HTco-localizedinthe
-HTtreatmentofNG2cellssignicantlyreducedtheexpressionofIL-1βandBDNFmRNAandincreasedthe
expressionofTNF-α.e5-HTreceptorspecicinhibitorsalverinecitrate,ketanserin,ondansetronandSB-399885blockedthe
regulatoryeectsof5-HTonNG2cells.ePLCsignalwaslinkedtothesecretionofIL-1β,TNF-αandBDNFinNG2cells.ese
resultsindicatedthat5-HTaectedIL-1β,TNF-α,andBDNFsecretionfromNG2cellsviathe5-HT1A,5-HT2A,5-HT3,5-HT6
receptorsandthePLCsignalingpathway.
[12].BDNFwasimplicatedintheregulationofneuro-
NG2gliacellsarewidespreadcellpopulationsthroughoutplasticity,synapticfunction,neuroprotectionandexcita-
grayandwhitematterinthecentralnervoussystem(CNS)tion/inhibitionimbalance[13].
andhavedistinctmorphologicalandphysiologicaltraits5-HTisanimportantmonoamineneurotransmitterin
[1–3].esecellsspecicallyexpressNG2chondroitintheCNS,anditissynthesizedprimarilyintheraphenuclei
sulfateproteoglycan(CSPG4)andplatelet-derivedgrowth[14].isneurotransmitterisabroadplayerinnumerous
factorreceptoralpha(PDGFRα)[4–6].eyareconsideredprocesses,inducingneuronalexcitabilityandmodulating
tobeanindependentpopulationofglialcells,otherwiseexcitatorysynaptictransmissionandsleep-wakebehavior
knownasoligodendrocyteprogenitorcells(OPCs)given[15,16].Serotonergicneuronsremostactivelyduring
thattheycandierentiateintooligodendrocytesduringwakefulness,reducetheirrateofactivityduringnon-rapid
development[7,8].eformationofdirectsynapticasso-eyemovementsleep(non-REMS),andaresilentduring
ciationsofNG2cellswithglutamatergicandc-amino-rapideyemovementsleep(REMS)[17].5-HTaectsthe
butylergicneuronsisavitalprocessinthetransmissionofdevelopment,proliferationanddierentiationofNG2cells.
informationintheCNS[9].Moreover,NG2cellsalsosecretItwasrecentlyreportedthatuoxetineincreased5-HT
somefactors,IL-1β,TNF-αandBDNF,whichaecttheinhibitsthebasalproliferationandsurvivalofOPCsinthe
activityofCNSneuronsandglialcells[10,11].Recentfornixandcorpuscallosumofadultmice[18].PLCisan
studiesrevealedthatIL-1βandTNF-αinuencedneuronalenzymethatmediatescellsignalingthroughvarious
2Evidence-BasedComplementaryandAlternativeMedicine
,theβ4serumfor30minutesat37°C,andtheliquidwasaspirated
subtypeisuniquelylocalizedinthegeniculatenucleusofthewithoutwashing,andthenincubatedwithprimaryantibodies
thalamus,whichispostulatedtohaveakeyroleinthefor2hoursat37°-
-β4−/−micebodiesincludedanti-NG2monoclonalantibodies(NG2-Ab,1:
exhibitedincreasedREMsleepandunusualwake-to-REM80,Abcam,USA)andanti-TPH(TPH-Ab,1:200,Abcam,
sleeptransitionsatnight[19].1eactionsofthe5-HT(2A),USA).Subsequently,sectionswereincubatedwithsecondary
5-HT(2B)and5-HT(2C)receptorsubtypesaremediatedbyantibodies,includingALexaFluor488(green,1:100)and
theactivationofPLC,resultingindepolarizationofthehostALexaFluor594(red,1:100,bothfromInvitrogen,USA),for1
cell[16].Ourpreviousstudyrevealedthatthedown-regu-hourat37°
lationof5-HTsynthesisviaparachlorophenylalaninecounterstainedwithaDAPIMix(BeijingSolebroTechnology
(PCPA),China).Finally,sectionswereobservedunderafluores-
Otherfunctionalrelationshipsbetween5-HTandNG2cellscenceinvertedmicroscope(Olympus,Japan).Fluorescence
mayexist,andfurtherstudiesshouldinvestigatetherelevantwasdetectedusingexcitationwavelengthsof488nm(green),
mechanismsof5-(red)and358nm(blue),(red
1epresentstudyexaminedwhetherNG2cellsecretionfluorescence)and5-HTneuronalmarkers(greenfluorescence)
ofIL-1β,TNF-αandBDNFwasassociatedwith5-
resultsshowedthattheeffectsof5-HTonNG2cellsecretionoverlappedinyellow(red+green=yellow),thetwomarkers
wasrelatedto5-HT1A,5-HT2A,5-HT3and5-HT6re-exhibitedco-expression[20].
ceptorsandthePLCsignalingpathway.
phase(2×10cells/well)wereseededin6-wellplatesand
-
(SD)rats()wereusedTNF-α,IL-1βandBDNFinthecellsupernatantswere
,accordingtothemanufacturer’s
-1β(ShenzhenXin
NationalInstitutesofHealthandinstitutionalguidelinesforBoshengBiotechnologyCo,China),TNF-α(ShenzhenXin
,China)andBDNF(Wuhan
thenumberofanimalsusedandminimizeanypainandElixiriteCorporation,China)[21].
discomfort.
-
,namelyratoli-growthphase(2×105cells/well)wereseededin6-well
godendrocyteprecursorcells(ROPCs),-
Qingqi(Shanghai,China)BiotechnologyDevelopmentCo,cellulartotalRNAwasextractedusingakit(Feiyang
’smodifiedBiologicalEngineeringCo,Guangzhou,China).cDNA
eaglemedium(DMEM)withhighglucose(Gibco,USA)wasreversetranscribedwithakit(FeiyangBiological
supplementedwith10%fetalbovineserum(FBS),penicillinEngineeringCo,Guangzhou,China),and2×SYBRGreen
(100U/ml)andstreptomycin(100µg/ml)at37°Cina5%qPCRMix(YisunBiotechnologyCo,Shanghai,China)was
--timePCR(StepOnePlus,
For5-HT-inducedNG2cellsecretionassays,accordingABICorporation,USA).Glyceraldehyde-3-phosphate
totheresultsofthepre-experiment,thecellsweretreateddehydrogenase(GAPDH)wasusedastheinternalref-
with5-HT(25,50and100μM)
treatedwithvariousreceptor(5-HT)%agarosegelelectrophoresisandvisualizedusing
activators/inhibitorsofintracellularsignals,suchas5-H1A,
5-HT2A,5-HT3and5-HT6receptors,includingthe5-experimentwasrepeatedthreetimeswiththreereplicate
−
HT1AR,5-HT2ARand5-HT6Rnonspecificantagonistwells,andtheresultswereanalyzedusingthe2ΔΔCt
asenapinemaleate(Ase,),the5-HT1Aspecificre-
ceptorinhibitoralverinecitrate(),the5-HT2Aandtherelatedsequencesusedintheexperimentsare
specificreceptorinhibitorketanserin(),the5-HT3showninTable1.
specificreceptorinhibitorondansetron(5μM),the5-HT6
specificreceptorinhibitorSB-399885(1μM),thePLCspe-
cificactivatorm-3M3FBS(3μM)
×5
U-73122().1esereagentswereappliedwith5-HTgrowthphase(210cells/well)wereseededin6-well
(100μM),andalltheexperimentalgroupswereincubatedfor
(Abclonal,
China),IL-1β(CellSignaling,China),TNF-α(CellSig-
-,China)andBDNF(Bioss,China)wasdetermined
removedafterperfusionandtheprefrontalcortexwascutusingWesternblot(WB)
into30-%goatseparatedusing5%gumconcentrateand12%separating
Evidence-BasedComplementaryandAlternativeMedicine3
Table1:Primersequencesusedinthisstudy.
PrimernameSequences(5ʹ-3ʹ)Productsize(bp)
ForwardAAGGACCACGGCTACACCATCTAC
5-HT1AR108
ReverseCTGACAGTCTTGCGGATTCGGAAG
ForwardCTTCCAACGGTCCATCCACA
5-HT2AR132
ReverseGGGCACCACATTACAACAAACAG
ForwardACATTTCCCTGTGGCGAACA
5-HT3R189
ReverseCAGTGGTTTCCCATGGCTGAG
ForwardCGCTGTGCTCCTGGGATAT
5-HT5R78
ReverseCCTGTTGAACGCCGTGTAGAT
ForwardGCACGAACTGGGCAAAGCT
5-HT6R86
ReverseGGACGCCACGAGGACAAAA
ForwardTTCTGTCGGTCTGGCTGCTCTC
5-HT7R130
ReverseCCGCAGTGGAGTAGATCGTGTAG
ForwardTGACAGGCAACCACTTACC
IL-1β123
ReverseCCCATACACACGGACAACT
ForwardGAAACAGTCTGCGAGGTGTG
TNF-α158
ReverseTTCTTCTTGCAGCCACACAC
ForwardTCTACGAGACCAAGTGTAATCCCAT
BDNF166
ReverseGAAGTGTCTATCCTTATGAACCGC
ForwardCTGAGGGCTCACGCAAGAA
PLCβ1102
ReverseGCAGCACGGTATAGGTGAA
ForwardACAGCAACAGGGTGGTGGAC
GAPDH98
ReverseTTTGAGGGTGCAGCGAACTTT
membraneswereblockedwithTBSTbufferandkeptatand5-HT.
-
spondingprimaryantibodywasdilutedintheblocking
-HT3,5-HT5,5-HT6,5-HT7Receptorsand
primaryantibodyincubationsolutionovernightat4°-HT1A,5--
1emembraneswererinsedthreetimeswithTBSTbuffertigatethefunctionalrelationshipbetweenNG2cellsand5-
andincubatedwithasecondaryAbfor2hoursatroomHT,real-timePCRwasusedtodetect5-HTreceptors
,-HT1A,5-
,5-HT3,5-HT5,5-HT6and5-HT7receptorswas
detectedonNG2cells(Figure2).
--HTInducedIL-1β,TNF-αandBDNFSecretionfrom
ticalanalysis().-1β,
means±-TNF-αandBDNFinNG2cellswasassociatedwith5-HT,
tinctionsbetweengroupswereanalyzedusingone-wayweuseddifferentconcentrationsof5-HT(25,50,and
analysisofvariance(one-wayANOVA)followedbyposthoc100μM)-HT
testsusingLSDorDunnett’-1βandBDNF
∗∗∗
p<,andp<-
-HTinducedthesecretionofIL-1β,TNF-α
andBDNFinNG2cells.
-HTModulatesNG2CellsSecretionofIL-1β,TNF-αand
-HTNeuronalMarkersBDNFvia5-HT1AR,5-HT2AR,5-HT3Rand5-HT6R.
(TPH)-HTreceptortypesonNG2cells.
cellsco-localizedwith5-HTneuronalmarkers(TPHs)inthe1erefore,weinvestigatedthereceptorsinvolvedinIL-1β,
prefrontalcortex,doubleimmunofluorescencestainingTNF--
(Ase)toblock5-HT1AR,5-
inFigure1,NG2cellsignalswereidentifiedasgreenHT2ARand5-HT6RandfoundelevatedsecretionofIL-1β,
fluorescence(Figure1(a)),and5-HTneuronmarker(TPH)TNF-αandBDNFinNG2cells(Figure4).Weusedspecific
sig